Complexes of peptide-binding fragments of heat shock proteins and their use as immunotherapeutic agents

Abstract

The present invention relates to pharmaceutical compositions comprising peptide-binding fragments of heat shock proteins (HSPs) and noncovalent complexes of peptide-binding fragments of HSPs in noncovalent association with antigenic molecules. The invention further relates to methods for the use of such pharmaceutical compositions as immunotherapeutic agents for the treatment and prevention of infectious diseases and cancer.

What is claimed is:  
     
         1 . A pharmaceutical composition comprising an amount of a molecular complex effective for treatment or prevention of an infectious disease or cancer, and a pharmaceutically acceptable carrier, said molecular complex comprising a heat shock protein peptide-binding fragment noncovalently associated with an antigenic molecule, said heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, and wherein said antigenic molecule displays the antigenicity of an antigen of an infectious agent or of a cancer cell.  
     
     
         2 . The pharmaceutical composition of    claim 1    wherein the heat shock protein is Hsp70, Hsp90, gp96, calreticulin, or PDI.  
     
     
         3 . The pharmaceutical composition of    claim 2    wherein the heat shock protein is Hsp70.  
     
     
         4 . The pharmaceutical composition of    claim 2    wherein the heat shock protein is Hsp90.  
     
     
         5 . The pharmaceutical composition of    claim 2    wherein the heat shock protein is PDI.  
     
     
         6 . The pharmaceutical composition of    claim 2    wherein the heat shock protein is gp96.  
     
     
         7 . The pharmaceutical composition of    claim 1    wherein the molecular complex is purified.  
     
     
         8 . The pharmaceutical composition of    claim 1    wherein said heat shock protein fragment lacks one or more other domains of the heat shock protein.  
     
     
         9 . A recombinant cell infected with a pathogen and transformed with a nucleic acid comprising a nucleotide sequence that (i) is operably linked to a promoter, and (ii) encodes a heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, which heat shock protein peptide-binding fragment noncovalently associates with an antigenic molecule when said antigenic molecule is present, to form a complex that in sufficient amount is capable of eliciting an immune response to the antigenic molecule.  
     
     
         10 . The recombinant cell of    claim 9    wherein said cell is a human cell.  
     
     
         11 . A recombinant cancer cell transformed with a nucleic acid comprising a nucleotide sequence that (i) is operably linked to a promoter, and (ii) encodes a heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, which heat shock protein peptide-binding fragment noncovalently associates with an antigenic molecule when said antigenic molecule is present, to form a complex that in sufficient amount is capable of eliciting an immune response to the antigenic molecule.  
     
     
         12 . The recombinant cancer cell of    claim 9    or    11    wherein the cell is a human cell.  
     
     
         13 . A recombinant cell transformed with (i) a first nucleic acid comprising a first nucleotide sequence that is operably linked to a first promoter and that encodes a heat shock protein peptide-binding fragment a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, and (ii) a second nucleic acid comprising a second nucleotide sequence that is operably linked to a second promoter and encodes an antigenic molecule, such that the heat shock protein peptide-binding fragment and the antigenic molecule are expressed within the cell and noncovalently associate with each other to form a complex that in sufficient amount is capable of eliciting an immune response to the antigenic molecule.  
     
     
         14 . A pharmaceutical composition comprising the recombinant cell of    claim 13    wherein said heat shock protein fragment lacks one or more other domains of the heat shock protein.  
     
     
         15 . A pharmaceutical composition comprising the recombinant cell of any one of claims  9 ,  11 , or  13  and a pharmaceutically acceptable carrier.  
     
     
         16 . A method for preparing a complex of a heat shock protein peptide-binding fragment noncovalently associated with a peptide, said heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, comprising: 
 a) culturing cells, transformed with a nucleic acid comprising a nucleotide sequence encoding the heat shock protein peptide-binding fragment and operably linked to a promoter, under conditions such that the heat shock protein peptide-binding fragment is expressed by the cells and associates with peptides of the cells; and    b) recovering a population of complexes of the heat shock protein peptide-binding fragment noncovalently associated with peptides from the cells.    
     
     
         17 . A method for preparing a heat shock protein peptide-binding fragment noncovalently associated with peptides derived from one or more antigens of an infectious agent, said heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-termninal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-termninal side with y number of amino acids that naturally flank the peptide-binding domain on the C-termninal side, wherein x plus y is not more than 400, comprising: 
 a) culturing infected cells, transformed with a nucleic acid comprising a nucleotide sequence encoding the heat shock protein peptide-binding fragment and operably linked to a promoter, under conditions such that the heat shock protein peptide-binding fragment is expressed by the cells and associates with peptides of the cells; and b) recovering from the cells a population of complexes of the heat shock protein peptide-binding fragment noncovalently associated with peptides derived from the infectious agent.    
     
     
         18 . A method for preparing a complex of a heat shock protein peptide-binding fragment noncovalently associated with a peptide, said heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, said method comprising digesting a preparation of heat shock proteins noncovalently associated with peptides with a protease under conditions and for a length of time sufficient for the formation of peptide-binding fragments of the heat shock protein noncovalently associated with peptides.  
     
     
         19 . A method for preparing a complex of a heat shock protein peptide-binding fragment noncovalently associated with a peptide, said heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, said method comprising: 
 a) digesting a preparation of heat shock proteins with a protease under conditions and for a length of time sufficient for the formation of peptide-binding fragments of the heat shock protein; and    b) contacting the peptide-binding fragments with peptides under conditions and for a length of time sufficient for the formation of complexes of heat shock protein peptide-binding fragments noncovalently associated with peptides.    
     
     
         20 . The method of any one of claims  16 - 19 , further comprising purifying the complexes.  
     
     
         21 . The method of any one of claims  16 - 19 , further comprising purifying the complexes by affinity chromatography.  
     
     
         22 . A method for preparing in vitro complexes of heat shock protein peptide-binding fragments noncovalently associated with one or more antigenic molecules, said heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, said method comprising incubating a heat shock protein peptide-binding fragment and one or more antigenic molecules under conditions and for a length of time sufficient for the formation of the complexes.  
     
     
         23 . The method of    claim 22    wherein the one or more antigenic molecules is a population of peptides from an infected cell or a cancer cell.  
     
     
         24 . The method of    claim 22    wherein the one or more antigenic molecules displays the antigenicity of an antigen of an infectious agent or a cancer cell.  
     
     
         25 . A method of eliciting an immune response against an antigen in an individual comprising administering to the individual an immunogenic complex of a heat shock protein peptide-binding fragment noncovalently associated with a first antigenic molecule displaying antigenicity of the antigen, said heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400.  
     
     
         26 . The method of    claim 25   , wherein said heat shock protein fragment lacks one or more other domains of the heat shock protein.  
     
     
         27 . The method of    claim 25   , further comprising, before, concurrently, or after administration of the immunogenic complex, administering to the individual a composition comprising antigen presenting cells sensitized in vitro with a sensitizing amount of a second immunogenic complex consisting essentially of a heat shock protein, or peptide-binding fragment thereof, noncovalently bound to a second antigenic molecule, in which said second antigenic molecule shares at least one antigenic determinant with the first antigenic molecule.  
     
     
         28 . A method of treating or preventing an infectious disease in an individual having an infectious disease, or in whom prevention of an infectious disease is desired, comprising administering to the individual an immunogenic complex of a heat shock protein peptide-binding fragment noncovalently associated with a first antigenic molecule, said heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, wherein the first antigenic molecule displays the antigenicity of an antigen of an infectious agent of the infectious disease.  
     
     
         29 . The method of    claim 28   , wherein said heat shock protein fragment lacks one or more other domains of the heat shock protein.  
     
     
         30 . The method of    claim 28   , further comprising, before, concurrently or after administration of the immunogenic complex, administering to the individual a composition comprising antigen presenting cells sensitized in vitro with a sensitizing amount of a second complex of a heat shock protein or peptide-binding fragment thereof noncovalently bound to a second antigenic molecule, said second antigenic molecule sharing at least one antigenic determinant with the first antigenic molecule.  
     
     
         31 . A method of treating or preventing an infectious disease in a subject having an infectious disease or in whom prevention of an infectious disease is desired comprising: 
 a) culturing an infected cell transformed with a nucleic acid comprising a nucleotide sequence encoding a heat shock protein peptide-binding fragment, said infected cell displaying the antigenicity of an antigen of an infectious agent of the infectious disease, said heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, said nucleotide sequence being operably linked to a promoter, under conditions such that the peptide-binding fragment is expressed by the infected cells and associates with peptides of the cell;    b) recovering complexes of the heat shock protein peptide-binding fragments noncovalently associated with peptides from the infected cell; and    c) administering to the subject an amount of the recovered complexes effective to treat or prevent the infectious disease.    
     
     
         32 . The method of    claim 31   , wherein said heat shock protein fragment lacks one or more other domains of the heat shock protein.  
     
     
         33 . The method of    claim 31   , further comprising, prior to step (a), the step of obtaining infected cells from the subject and transforming the infected cells with the nucleic acid.  
     
     
         34 . The method of    claim 31   , further comprising, prior to step (a), the step of obtaining the infected cell from one or more individuals and transforming the infected cells with the nucleic acid, said one or more individuals being different from the subject and having the same type of infectious disease as the subject.  
     
     
         35 . A method of treating or preventing an infectious disease in a subject having an infectious disease or in whom prevention of an infectious disease is desired comprising: 
 a) culturing a recombinant cell transformed with (i) a first nucleic acid encoding a heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, and (ii) a second nucleic acid encoding an antigenic molecule displaying the antigenicity of an antigen of an infectious agent of the infectious disease;    b) recovering complexes of the heat shock protein peptide-binding fragments noncovalently associated with the antigenic molecule; and    c) administering to the subject an amount of the recovered complexes effective to treat or prevent the infectious disease.    
     
     
         36 . The method of    claim 28   ,    31   , or  35 , in which the infectious disease is caused by an infectious agent selected from the group consisting of a virus, a bacterium, a fungus, and a parasite.  
     
     
         37 . A method of treating or preventing cancer in an individual having a type of cancer or in whom prevention of a type of cancer is desired comprising administering to the individual an immunogenic complex of a heat shock protein peptide-binding fragment noncovalently associated with a first antigenic molecule, said heat shock protein comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, wherein either (a) the first antigenic molecule displays antigenicity of said type of cancer or a metastasis thereof; or (b) the complex is obtained by recovering complexes from said type of cancer cells or a metastasis thereof that recombinantly express the heat shock protein peptide-binding fragment.  
     
     
         38 . The method of    claim 37   , further comprising, before, concurrently or after administration of the immunogenic complex, administering to the individual a composition comprising antigen presenting cells sensitized in vitro with a sensitizing amount of a second complex of a heat shock protein or peptide-binding fragment thereof noncovalently bound to a second antigenic molecule, said second antigenic molecule sharing at least one antigenic determinant with the first antigenic molecule.  
     
     
         39 . A method of treating or preventing cancer in a subject having a type of cancer or in whom prevention of a type of cancer is desired comprising: 
 a) culturing a cancer cell transformed with a nucleic acid comprising a nucleotide sequence encoding a heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, said nucleotide sequence being operably linked to a promoter, under conditions such that the peptide-binding fragment is expressed by the cancer cell and associates with peptides of the cell;    b) recovering complexes of the heat shock protein peptide-binding fragments noncovalently associated with peptides from the cancer cell; and    c) administering to the subject an amount of the recovered complexes effective to treat or prevent cancer.    
     
     
         40 . The method of    claim 39   , further comprising, prior to step (a), the step of obtaining cancer cells from the subject and transforming the cancer cells with the nucleic acid.  
     
     
         41 . The method of    claim 39   , further comprising, prior to step (a), the step of obtaining cancer cells from one or more individuals and transforming the cancer cells with the nucleic acid, said one or more individuals being different from the subject and having the same type of cancer as the subject.  
     
     
         42 . A method of treating or preventing cancer in a subject having a type of cancer or in whom prevention of a type of cancer is desired comprising: 
 a) culturing a recombinant cell transformed with (i) a first nucleic acid encoding a heat shock protein peptide-binding fragment comprising a peptide-binding domain that is contiguous on the N-terminal side with x number of amino acids that naturally flank the peptide-binding domain on the N-terminal side, and that is contiguous on the C-terminal side with y number of amino acids that naturally flank the peptide-binding domain on the C-terminal side, wherein x plus y is not more than 400, and (ii) a second nucleic acid encoding an antigenic molecule displaying the antigenicity of an antigen of a cancer cell;    b) recovering complexes of the heat shock protein peptide-binding fragments noncovalently associated with the antigenic molecule; and    c) administering to the subject an amount of the recovered complexes effective to treat or prevent cancer.    
     
     
         43 . The method of    claim 25   ,    28   ,  31 ,  35 ,  37 ,  39 , or  42 , wherein the peptide-binding domain consists of the peptide-binding domain of Hsp70, Hsp90, gp96, calreticulin, PDI, or a mixture of two or more of the foregoing.  
     
     
         44 . The method of    claim 43   , wherein the heat shock protein peptide-binding fragment is a human gp96 peptide-binding fragment comprising the amino acid sequence from about position 5 to about position 232 of the amino acid sequence shown in FIG. 2C (SEQ ID NO:7).  
     
     
         45 . The method of    claim 43   , wherein the heat shock protein peptide-binding fragment is a gp96 peptide-binding fragment comprising the amino acid sequence from about position 615 to about position 658 of the amino acid sequence shown in FIG. 2C (SEQ ID NO:7).  
     
     
         46 . The method of    claim 43   , wherein the heat shock protein peptide-binding fragment is a gp96 peptide-binding fragment comprising the amino acid sequence from about position 624 to about position 630 of the amino acid sequence shown in FIG. 2C (SEQ ID NO:7).